Promoting the Synthesis of Precursor Substances by Overexpressing Hexokinase (Hxk) and Hydroxymethylglutaryl-CoA Synthase (Erg13) to Elevate ß-Carotene Production in Engineered Yarrowia lipolytica.

As a valuable carotenoid, ß-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of ß-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and ß-carotene biosynthesis, this study aims to increase the ß-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered Yarrowia lipolytica. In this study, we investigated the effect of the unique hexokinase gene (Hxk) overexpression on ß-carotene accumulation and glucose consumption. The Hxk gene was introduced into a ß-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the ß-carotene content by 98%. Overexpression of the Hxk gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of Hxk (315% higher) compared to the control Y.L-1 strain. Moreover, Hxk overexpression accelerated the utilization rate of glucose. The gene erg13 encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for ß-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of erg13 produced 8.41 mg/g dry cell weight (DCW) of ß-carotene, which was 259% higher than Y.L-1. The ß-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating erg13 into the chromosome and Hxk overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the erg13 gene. Finally, the titer of ß-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of Hxk and erg13 could improve ß-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of ß-carotene synthesis.

[Comparison of the desulfurization activity among several bacteria and analysis of the conservation of their desulfurization genes].

Several bacteria, Bacillus brevis R-6, Pseudomonas delafleldii R-8, Nocardia globerula R-9, Bacillus sphaericus R-16, Rhodococcus erythropolis LSSE8-1 and Gordonia nitida LSSEJ-1, which can convert dibenzothiophene into 2-hydroxybiphenyl and sulfate, were investigated. Desulfurization products were quantitively determined by HPLC. Result revealed that each of these bacteria desulfurize DBT at a different rate. In order to obtain more information, the fragments encoding desulfurizing enzymes were studied. Desulfurization genes of R-6 and R-8 were separately amplified via PCR with specific primers based on the related sequences of Rhodococcus sp. IGTS8. Both sequences areminimally 99% related to IGTS8 sequence. Afterwards, dsz operon of LSSEJ-1 and R-9 were amplified and cloned. Sequences are also highly conservative. Data shows that identity of dszA between R-9 and IGTS8 is 99.6%, and identity of dszA between LSSEJ-1 and IGTS8 is 99.9%; dszB sequence of R-9 and LSSEJ-1 is 99.6% similarity to their counterpart sequence from IGTS8;Identity of dszC between R-9 and IGTS8 is 99.9%, and identity of dszC between LSSEJ-1 and IGTS8 is 99.1% . It may be deduced that the origins of desulfurization genes from mesophilic bacteria are the same.