Highlights d A genetic circuit designed to functionally select anti-CRISPR genes from metagenomes d Four protein families, AcrIIA7-10, that inhibit Cas9 in vivo and in vitro were identified d AcrIIA7-10 are widely distributed across seven bacterial phyla d Dissemination patterns of AcrIIA7-10 suggest interphylum horizontal gene transfer
Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.
Genome editing using CRISPR/Cas9 was successfully demonstrated in Esherichia coli to effectively produce n-butanol in a defined medium under microaerobic condition. The butanol synthetic pathway genes including those encoding oxygen-tolerant alcohol dehydrogenase were overexpressed in metabolically engineered E. coli, resulting in 0.82 g/L butanol production. To increase butanol production, carbon flux from acetyl-CoA to citric acid cycle should be redirected to acetoacetyl-CoA. For this purpose, the 5'-untranslated region sequence of gltA encoding citrate synthase was designed using an expression prediction program, UTR designer, and modified using the CRISPR/Cas9 genome editing method to reduce its expression level. E. coli strains with decreased citrate synthase expression produced more butanol and the citrate synthase activity was correlated with butanol production. These results demonstrate that redistributing carbon flux using genome editing is an efficient engineering tool for metabolite overproduction.
S. cerevisiae sake K6 was the firstly isolated industrial strain to overproduce S-adenosyl-L-methionine (SAM). Although the strain has advantages over other strains, such as GRAS (generally recognized as safe) property, the S. cerevisiae K6 has not been further developed with DNA recombinant technology due to the lack of a proper genetic marker. In this study, UV mutagenesis was conducted with S. cerevisiae sake K6. With the method, a mutant of sake yeast with leucine auxotroph, K6-1, was isolated. The mutant showed comparable growth rate and SAM productivity with its wild type. Using the auxotroph as a genetic marker, a SAM synthase in S. cerevisiae, SAM2, was overexpressed in the mutant strain. This recombinant DNA technology successfully increased SAM productivity in sake yeast.
Engineered microbes
often suffer from reduced fitness resulting
from metabolic burden and various stresses. The productive lifetime
of a bioreactor with engineered microbes is therefore susceptible
to the rise of nonproductive mutants with better fitness. Synthetic
addiction is emerging as a concept to artificially couple the growth
rate of the microbe to production to tackle this problem. However,
only a few successful cases of synthetic addiction systems have been
reported to date. To understand the limitations and design constraints
in long-term cultivations, we designed and studied conditional synthetic
addiction circuits in Saccharomyces cerevisiae. This
allowed us to probe a range of selective pressure strengths and identify
the optimal balance between circuit stability and production-to-growth
coupling. In the optimal balance, the productive lifetime was greatly
extended compared with suboptimal circuit tuning. With a too-high
or -low pressure, we found that production declines mainly through
homologous recombination. These principles of trade-off in the design
of synthetic addition systems should lead to the better control of
bioprocess performance.
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