Microbial Synthetic Biology, Systems Metabolic Engineering and Enzyme Engineering for Advanced Microbial Biodiesel Production with Saccharomyces cerevisiae

The continuous requirement of transportation biofuels has brought the necessity to establish alternatives permitting low-cost production of biodiesel while being environmentally friendly. Biodiesel production was achieved utilizing Saccharomyces cerevisiae by employing respective enzymes that catalyze the synthesis of fatty acid ethyl esters (FAEEs) based on fatty acyl-CoA molecules and ethanol. Five acyltransferases/wax ester synthases were tested and heterologously introduced in yeast by expressing their codon-optimized gene for expression in a yeast host under the strong promoter TEF1p using plasmid pSP-GM2. In conclusion MhWS2 from oil bacteria Marinobacter hydrocarbonoclasticus was the highliest active with 8.1 pmol/(mg protein•min).Through Metabolically Engineering, metabolism was widely modified for increasing biodiesel production by eliminating fatty acid-consumption competitive pathways, therefore augmenting the fatty acid pool. This was achieved by deleting genes ARE1, ARE2, LRO1, DGA1 and POX1, which conferred a 5-fold increase of FAEEs formation (17.2 mg/L). Right after, MhWS2 was overproduced in yeast by chromosomal integration of its codon-optimized ws2. Then gene copy number was enhanced by integrating it in δ-regions, conferring 7.5-fold higher biodiesel production in a gradually evolved strain tolerant to 20 mg/mL antibiotic G418.Furthermore, Protein Engineering of two natural catalysts (MhWS2 and α/β-hydrolase Eeb1p homolog to yeast Saccharomyces cerevisiae) was addressed. In these subprojects, directed evolution of these two enzymes was achieved for favoring the synthesis of biodiesel by augmenting their efficiency and altering selectivity towards biocatalyzing FAEEs of desired chain length (C16 and C18, either saturated or monounsaturated). Starting with random mutagenesis of the respective codifying genes (ws2 and EEB1) allowed libraries of random point-mutations. Then library screening was conducted for reducing the CFU (colony formation unit) number; since lipotoxicity was employed as screening method due the condition of the yeast mutants, modifying to a weaker promoter was needed: KEX2p was then further applied. Ultimate selection of the best evolved variants of these enzymes was performed: variants MhWS2-v11 (65.3%) increment when compared to natural MhWS2) and Eeb1p-v04 (45.7% increment). MhWS2-v11 possesses five residue substitutions, while Eeb1p-v04 has 19 residue substitutions. In this case of scientific and technological studies, an advanced biofuel of an upcoming generation has been produced

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity > 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae

Abstract: Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. Key Points: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity

Prospects for microbial biodiesel production

As the demand for biofuels for transportation is increasing, it is necessary to develop technologies that will allow for low-cost production of biodiesel. Conventional biodiesel is mainly produced from vegetable oil by chemical transesterification. This production, however, has relatively low land-yield and is competing for agricultural land that can be used for food production. Therefore, there is an increasing interest in developing microbial fermentation processes for production of biodiesel as this will allow for the use of a wide range of raw-materials, including sugar cane, corn, and biomass. Production of biodiesel by microbial fermentation can be divided into two different approaches, (1) indirect biodiesel production from oleaginous microbes by in vitro transesterification, and (2) direct biodiesel production from redesigned cell factories. This work reviews both microbial approaches for renewable biodiesel production and evaluates the existing challenges in these two strategies

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid ethyl esters, an advanced biofuel, by eliminating non-essential fatty acid utilization pathways

Microbial production of fatty acid derived chemicals and fuels is currently of great interest due to the limited resources and increasing prices of petroleum and petroleum-based products. The development of Saccharomyces cerevisiae as a fatty acid ethyl ester (FAEE) cell factory would represent an opportunity for biodiesel production due to its successful history in the biotechnology area. However, fatty acid (FA) biosynthesis is highly regulated and usually not high enough for developing an efficient production process. In S. cerevisiae, FAs are degraded by beta-oxidation and a large fraction is utilized to synthesize steryl esters (SEs) and triacylglycerols (TAGs), which are not essential for the cell. Here, by eliminating nonessential FA utilization pathways, we developed a metabolic engineering strategy resulting in a S. cerevisiae strain that can overproduce FAs and in turn use these for producing FAEEs (biodiesel). Compared to the wild-type, there is an about 3-fold increase in free FA content in a strain devoid of both TAG and SE formation, a 4-fold increase in free FA content in a strain that is incapable of beta-oxidation, and a 5-fold increase of free FAs in a strain lacking all of these non-essential FA utilization pathways. It is also demonstrated that there are similar positive effects on FAEE production in these deletion strains. The highest production of FAEEs is 17.2 mg/l in the strain in which all these pathways were blocked. The results of this study serve as a basis for further strategies to improve the production of FA derivatives in S. cerevisiae. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved

Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration

Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms\u27 chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs(SE)(L641P)) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1(S1157A,) (S659A)) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs(SE)(L641P), ACC1(S1157A, S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1 Delta, are2 Delta, dga1 Delta and lro1 Delta) and beta-oxidation (pox1 Delta) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae

Improvement on the productivity of continuous tequila fermentation by Saccharomyces cerevisiae of Agave tequilana juice with supplementation of yeast extract and aeration

Agave (Agave tequilana Weber var. azul) fermentations are traditionally carried out employing batch systems in the process of tequila manufacturing; nevertheless, continuous cultures could be an attractive technological alternative to increase productivity and efficiency of sugar to ethanol conversion. However, agave juice (used as a culture medium) has nutritional deficiencies that limit the implementation of yeast continuous fermentations, resulting in high residual sugars and low fermentative rates. In this work, fermentations of agave juice using Saccharomyces cerevisiae were put into operation to prove the necessity of supplementing yeast extract, in order to alleviate nutritional deficiencies of agave juice. Furthermore, continuous fermentations were performed at two different aeration flow rates, and feeding sterilized and non-sterilized media. The obtained fermented musts were subsequently distilled to obtain tequila and the preference level was compared against two commercial tequilas, according to a sensorial analysis. The supplementation of agave juice with air and yeast extract augmented the fermentative capacity of S. cerevisiae S1 and the ethanol productivities, compared to those continuous fermentations non supplemented. In fact, aeration improved ethanol production from 37 to 40 g L-1, reducing sugars consumption from 73 to 88 g L-1 and ethanol productivity from 3.0 to 3.2 g (Lh)(-1), for non-aerated and aerated (at 0.02 vvm) cultures, respectively. Supplementation of yeast extract allowed an increase in specific growth rate and dilution rates (0.12 h(-1), compared to 0.08 h(-1) of non-supplemented cultures), ethanol production (47 g L-1), reducing sugars consumption (93 g L-1) and ethanol productivity [5.6 g (Lh)(-1)] were reached. Additionally, the effect of feeding sterilized or non-sterilized medium to the continuous cultures was compared, finding no significant differences between both types of cultures. The overall effect of adding yeast extract and air to the continuous fermentations resulted in 88 % increase in ethanol productivity. For all cultures, pH was not controlled, reaching low pH values (from 2.6 to 3). This feature suggested a reduced probability of contamination for prolonged continuous cultures and explained why no significant differences were found between continuous cultures fed with sterilized or non-sterilized media. Concentrations of volatile compounds quantified in the distillates (tequila) were in the allowed ranges established by the Mexican regulation of tequila (NOM-006-SCFI-2012, Norma Oficial Mexicana: Bebidas alcoholicas-Tequila-specificaciones, 2012). The preference level of the distillates was similar to that of two well-known commercial tequilas. The results suggested the possibility of implementing this innovative technology on an industrial scale, attaining high productivities and using non-sterilized agave juice

Improvement on the productivity of continuous tequila fermentation by Saccharomyces cerevisiae of Agave tequilana juice with supplementation of yeast extract and aeration

Agave (Agave tequilana Weber var. azul) fermentations are traditionally carried out employing batch systems in the process of tequila manufacturing; nevertheless, continuous cultures could be an attractive technological alternative to increase productivity and efficiency of sugar to ethanol conversion. However, agave juice (used as a culture medium) has nutritional deficiencies that limit the implementation of yeast continuous fermentations, resulting in high residual sugars and low fermentative rates. In this work, fermentations of agave juice using Saccharomyces cerevisiae were put into operation to prove the necessity of supplementing yeast extract, in order to alleviate nutritional deficiencies of agave juice. Furthermore, continuous fermentations were performed at two different aeration flow rates, and feeding sterilized and non-sterilized media. The obtained fermented musts were subsequently distilled to obtain tequila and the preference level was compared against two commercial tequilas, according to a sensorial analysis. The supplementation of agave juice with air and yeast extract augmented the fermentative capacity of S. cerevisiae S1 and the ethanol productivities, compared to those continuous fermentations non supplemented. In fact, aeration improved ethanol production from 37 to 40 g L-1, reducing sugars consumption from 73 to 88 g L-1 and ethanol productivity from 3.0 to 3.2 g (Lh)(-1), for non-aerated and aerated (at 0.02 vvm) cultures, respectively. Supplementation of yeast extract allowed an increase in specific growth rate and dilution rates (0.12 h(-1), compared to 0.08 h(-1) of non-supplemented cultures), ethanol production (47 g L-1), reducing sugars consumption (93 g L-1) and ethanol productivity [5.6 g (Lh)(-1)] were reached. Additionally, the effect of feeding sterilized or non-sterilized medium to the continuous cultures was compared, finding no significant differences between both types of cultures. The overall effect of adding yeast extract and air to the continuous fermentations resulted in 88 % increase in ethanol productivity. For all cultures, pH was not controlled, reaching low pH values (from 2.6 to 3). This feature suggested a reduced probability of contamination for prolonged continuous cultures and explained why no significant differences were found between continuous cultures fed with sterilized or non-sterilized media. Concentrations of volatile compounds quantified in the distillates (tequila) were in the allowed ranges established by the Mexican regulation of tequila (NOM-006-SCFI-2012, Norma Oficial Mexicana: Bebidas alcoholicas-Tequila-specificaciones, 2012). The preference level of the distillates was similar to that of two well-known commercial tequilas. The results suggested the possibility of implementing this innovative technology on an industrial scale, attaining high productivities and using non-sterilized agave juice